The present invention relates to an objective unit which is for use in an optical microscope and which performs incident lighting, and more particularly to an objective unit which uses a space outside the observation light path to perform lighting for accomplishing dark-field observation or for generating evanescent waves to achieve incident-light fluorescent observation.
FIG. 7 is a sectional view of the illumination system of an optical microscope. The microscope comprises an objective unit 3, a focusing lens 4, and eye lens (not shown), a lamp 7, a collector lens 9, and mirror 10. To obtain a magnified image of a sample 1 (i.e., an object of observation), the objective unit 3, focusing lens 4 and eye lens are arranged in the axis 2 of observation light. The objective unit 3 contains an objective lens system 6.
The lamp 7 is located at a predetermined distance from the observation-light axis 2. The lamp 7 emits light for incident lighting, which has an illumination-light axis 8 extending at right angles to the observation-light axis 2. Arranged in the illumination-light axis 8 are the collector lens 9 and the mirror 10. The collector lens 9 is semi-spherical, while the mirror 10 is shaped like a ring. The mirror 10 is inclined at 45.degree. to the observation-light axis 2 and the illumination-light axis 8. The mirror 10 has an annular reflecting surface.
The collector lens 9 collects the light emitted from the lamp 7 for incident lighting, converting the light to a light beam having a circular cross section. The light beam reaches the mirror 10. Part of the light beam passes through the opening of the mirror 10. The remaining part of the light beam is reflected downwards from the reflecting surface of the mirror 10. It is used as an illumination light (incident illumination light) 11 which is suitable for incident lighting. The light beam has an annular cross section and is coaxial with the observation-light axis 2. The illumination light 11 passes surrounding the observation light path and is guided into objective unit 3.
The illumination light 11 for incident lighting passes through the objective unit 3, surrounding the lens system 6. The illumination light 11 is then reflected by a mirror 12 and converged onto the sample 1. The observation light beam 5 reflected from the sample 1 passes through the lens system 6 of the unit 3. The light beam 5 then passes through the focusing lens 4. Eventually the light beam 5 is guided to the eye lens (not shown), whereby an observer can see a magnified image of the sample 1.
The objective unit 3 will be described in detail, with reference to FIG. 8 which is a sectional view. The objective unit 3 is a dry objective unit. As seen from FIG. 8, the unit 3 comprises lens-holding frames 20a to 20g, a hollow cylinder 21, and a lens-holding ring 22, besides the lens system 6. The lens system 6 consists of seven lenses 6a to 6g which are arranged in the observation-light axis 2. The lenses 6a to 6g are held by the lens-holding frames 20a to 20g, respectively. The frames 20a to 20g are provided in the hollow cylinder frame 21 and held in place once the lens-holding ring 22 has been fitted into one end of the hollow cylinder frame 21.
As shown in FIG. 8, a ring 25 is mounted on the objective unit 3. The ring 25 will be described with reference to FIG. 9, which is a part of a sectional view taken along line IX--IX in FIG. 8. As FIG. 9 shows, the ring 25 has two arcuate elongated holes 23 and 24. Thus, the ring has two parts in which no holes are made. The holes 23 and 24 are arranged in the same circle and separated from each other. The ring 25 has a screw thread 26 on its outer circumferential surface.
As seen from FIG. 8, a hollow cylindrical cover 27 is mounted on the mirror 12 and also on the ring 25. The cover 27 is set in screw engagement with the ring 25 at the screw 26 thereof, positioned coaxial with the hollow cylinder 21. Hence, an annular space is provided between the hollow cylinder 21 and the hollow cylindrical cover 27 by holes 23 and 24. The annular space serves as illumination light path. The mirror 12, which is fitted in the distal end of the cover 27, reflects the illumination light 11 at its reflecting surface 12a, applying the light 11 onto the sample 1.
As illustrated in FIG. 8, a cover glass 30 is located below the objective unit 3. The cover glass 30 has a sample-mounting surface 29. The hollow cylindrical cover 27 has a screw 27a on its proximal end. At the screw 27a the objective unit 3 is removably attached to the body of the microscope.
In the illumination system shown in FIG. 7, the lamp 7 is the illumination light source, and the illumination light 11 is applied into the objective unit 3 by using the collector lens 9 and the mirror 10.
The illumination light 11 is applied to the sample 1, with a numerical aperture (NA) larger than that of the observation light. The 0th-order light totally reflected from the sample 1 does not enter the observation light path provided in the objective unit 3. The high-order light diffracted and scattered from the sample 1 enters the lens 6a set in the observation light path and passes through the lens 6b to 6g set also in the observation light path. The high-order light is applied to the focusing lens 4, which focuses the light. The light thus focused is guided to the eye lens (not shown) or a video camera (not shown), etc. Dark-field observation of the sample 1 is thereby accomplished.
The objective lens 3 may be an oil immersion objective. In this case, assume that the cover glass 30, the oil and the sample 1 have refractive indices of 1.5, 1.5 and 1.3, respectively. Then, the illumination light applied with NA of 1.3 or more is totally reflected at the interface between the cover glass 30 and the sample 1. In this case, evanescent light is generated on the side of the sample 1. The evanescent light may be used as excitation light in the oil immersion objective unit. Fluorescent observation can then be achieved by use of the illumination path independent of the observation system even if the objective unit comprises lenses having great NAs. This is utilized when objective lenses designed for mono-molecular fluorescent observation are employed in order to reduce auto-fluorescent light and attain a high S/N ratio.
Being a dry system, not a liquid-immersion system, the objective unit 3 shown in FIG. 8 has a small NA and a long working distance (WD), i.e., the distance between its distal end and the cover glass 30. Therefore, the unit 3 has a sufficient space for the illumination light path. On the other hand, most liquid immersion objective units, particularly oil immersion ones, comprises lenses having a large NA of about 1 to about 1.45 and have a short working distance of about 0.1 mm. In such a liquid immersion objective unit, the distal end, the lens-holding frames and the hollow cylinder make it difficult to provide a space large enough for an illumination light path.
To enable the objective unit 3 to apply as much illumination light as possible to the sample, it is necessary to increase the NA of the illumination light 11. If the light 11 has an NA as large as that of the observation light beam, i.e. it has a large marginal ratio, the peripheral part of the light 11 will be blocked by the lens-holding frames. Hence, the illumination light 11 cannot have a large NA.
To increase the NA of the illumination light 11, the wall thickness of the lens-holding frames 20a to 20g may be decreased, making it possible to broaden the annular space for passing the illumination light 11. If the frames 20a to 20g have less wall thickness, however, they will become less rigid than is required to hold the lenses 6a to 6g in a completely coaxial relationship. And, it is difficult to ensure a high degree of precision with respect to the dimensions of the frames. In addition, the size of the objective unit 3 is limited; its overall length should be 45 mm or less. The lenses cannot be made thicker and the lens-holding frames cannot be made longer, in order to increase the working distance so as to provide a space sufficient for an illumination light path.